Lung Physiology.
The main passageways for air to travel from the nose or mouth to the lungs are the bronchi, which eventually branch to the bronchioles, which then branch to alveolar ducts. The terminal airspaces of the lungs, the alveoli, where gas exchange takes place, branch off of the alveolar ducts. Air pressure is equal between two adjacent alveoli. Thus, as equal pressures are applied to each side of any wall, or septum, between adjacent air-filled alveoli, such septa are substantially planar in shape. The surface of the alveolus is lined with type I and II alveolar epithelial cells, on top of which there is a thin liquid lining layer. Thus, there is an air-liquid interface in the lungs that has an associated surface tension. Alveolar type II epithelial cells release surfactant, which adsorbs to the interface and maintains low surface tension in the lungs. Lung surfactant is a mixture of phospholipids, the most abundant of which is dipalmitoylphosphatidylcholine (DPPC); neutral lipids; and four surfactant-associated proteins, surfactant protein (SP)-A, SP-B, SP-C and SP-D. Surfactant proteins B and C, which are hydrophobic, facilitate surfactant lipid adsorption. By lowering surface tension, surfactant reduces the pressure required to keep the lungs inflated and reduces the work of breathing.
Inside of alveolar septa are located the pulmonary capillaries. The tissue and liquid between capillary blood and alveolar air constitute the alveolar capillary barrier, across which gas exchange occurs.
Acute Respiratory Distress Syndrome (ARDS) and Ventilation-Induced Lung Injury.
The acute respiratory distress syndrome can be caused by any number of different initial insults. Regardless of cause, with ARDS there is inflammation in the lungs. With inflammation, there is increased permeability of the alveolar-capillary barrier and liquid leaks out of the blood vessels. The liquid carries with it plasma proteins—principally the most abundant plasma protein, albumin, but also plasma proteins present at lower concentrations, such as fibrinogen. When enough liquid escapes from the vessels, liquid begins to enter the alveoli, a condition known as alveolar edema. In flooded alveoli, the air-liquid interface forms a meniscus. Thus, as described by the Laplace relation, flooded alveolar liquid pressure is less than alveolar air pressure, to a degree that is proportional to surface tension at the meniscal interface. The additional liquid in the airspace effectively thickens the alveolar-capillary barrier across which gas exchange occurs.
Further, with alveolar edema, there are regions of the lungs in which alveolar flooding is heterogeneous. That is, aerated and liquid-flooded alveoli are interspersed. ‘Intervening’ septa, i.e., those located between adjacent aerated and flooded alveoli, are thus subjected to a relatively high air pressure on one side and a relatively low liquid pressure on the other. The air-liquid pressure difference across the intervening septum, which equals the pressure difference across the meniscus of the flooded alveolus and is proportional to surface tension, causes the intervening septum to bow into the flooded alveolus. Thus, at any given lung inflation pressure, the intervening septum is extended beyond its normal length and becomes a site of stress concentration.
Patients with ARDS are treated by mechanical ventilation, which assists gas exchange but often causes an over-distension injury that exacerbates the underlying lung disease and prevents patient recovery. In particular, mechanical ventilation inflates the lung above the volumes reached during spontaneous breathing, thus increasing surface tension above normal and, as a result, exacerbates the surface tension-dependent stress concentrations in intervening septa between aerated and flooded alveoli. Consequently, mechanical ventilation of heterogeneously flooded regions injuriously increases permeability of an initially intact alveolar-capillary barrier to a degree that is proportional to the surface tension of the alveolar liquid in the region.
As over-distension injury is surface tension-dependent, lowering surface tension of the liquid in the alveoli of an edematous lung should directly lessen ventilation injury. Clinical surfactant therapy trials have tested intratracheal instillation of exogenous animal (either bovine, e.g., SURVANTA® (commercially available from Abbvie, Inc. located in North Chicago, Ill., U.S.A.), or porcine) surfactant as a means of lowering surface tension and treating ARDS. SURVANTA® is intended to provide surface-tension lowering properties similar to that of natural (endogenous) lung surfactant and is generally a mixture of bovine-harvested phospholipids, including DPPC, neutral lipids, fatty acids and surfactant proteins B and C; additional DPPC; palmitic acid; and tripalmitin, all suspended in a 0.9% sodium chloride solution. More specifically, SURVANTA® typically has a phospholipid concentration of about 25 milligrams per milliliter (mg/mL), a triglyceride concentration of from about 0.5 mg/mL to about 1.75 mg/mL, and a protein content of less than about 1.0 mg/mL. However, such exogenous surfactant therapy has not reduced ARDS mortality. One possible reason for the failure of exogenous surfactant therapy is heterogeneity of exogenous surfactant distribution throughout the lungs.
Further, the heterogeneous alveolar flooding pattern is attributable to liquid being trapped in discrete alveoli by a ‘pressure barrier,’ i.e., the presence of a higher liquid pressure at the edge than in the center of flooded alveoli. And the pressure barrier is proportional to surface tension at the air-liquid interface. Lowering surface tension, by lowering the pressure barrier, can facilitate liquid escape from flooded alveoli and redistribution, in a more homogeneous fashion, across neighboring alveoli. Such liquid escape from flooded alveoli reduces alveolar flooding heterogeneity and should reduce the number of stress concentrations present. Thus lowering surface tension should also, by reducing flooding heterogeneity, indirectly reduce mechanical ventilation injury.
Cardiogenic Pulmonary Edema (CPE).
In cardiogenic pulmonary edema, liquid entrance into the alveoli of the lung is driven not by abnormally elevated permeability of the alveolar-capillary barrier but, rather, by abnormally elevated pulmonary capillary blood pressure secondary to left heart dysfunction. As barrier permeability is, at least initially, normal, plasma proteins should be trapped in the no capillaries and plasma protein concentration in the alveolar edema liquid should be low. Yet, quantitative analysis of alveolar liquid in CPE demonstrates that protein concentration is elevated above normal in CPE, to the same degree as in ARDS. Further, in CPE, as in ARDS, there are regions of the lungs in which alveolar flooding is heterogeneous.
It may be that mechanical ventilation of CPE patients exacerbates stress concentrations in regions of heterogeneous alveolar flooding, thus injuring the alveolar-capillary barrier in such regions and leading to plasma protein entrance into the edema liquid. Regardless of the mechanism responsible for the elevated edema liquid plasma protein concentration in CPE patients, however, alveolar flooding pattern and edema liquid plasma protein concentration are similar between CPE and ARDS. In CPE, as in ARDS, lowering surface tension should, by either direct or indirect means, lessen ventilation injury of regions with heterogeneous alveolar flooding.
Neonatal Respiratory Distress Syndrome (NRDS).
Lung surfactant is produced during the third trimester of gestation and is critical to the ability of a baby to breathe unaided. Historically, many premature babies did not survive. Since the 1980's, tracheal instillation of exogenous animal surfactant has been a tremendously successful therapy that has enabled premature babies to live. However, there remains room for improvement in the clinical treatment of NRDS.
As the lungs are entirely filled with liquid prior to the first breath following birth, there are similarities between neonatal and edematous lungs. Neonatal respiratory distress is similar to CPE, in particular, in that both barrier permeability and alveolar liquid protein concentration are, initially, normal/low. However, with mechanical ventilation, barrier permeability and alveolar liquid protein content increase. This increase is likely attributable to mechanical ventilation causing heterogeneous aeration, thus leaving behind heterogeneous flooding and resulting in exacerbation of stress concentrations in heterogeneously flooded regions. An important difference between NRDS and both ARDS and CPE is that in NRDS there is less surfactant present than in mature lungs.
In NRDS, lowering surface tension with exogenous surfactant therapy is already beneficial. However lowering surface tension to a greater degree, or more uniformly throughout the lungs, should further lessen ventilation injury of regions with heterogeneous flooding.
Surface Tension Assessment Methods.
Surface tension is assessed in the isolated lung and in vitro using four complementary methods, as follows.
Method 1. Surface tension determination in the adult rat lung. In the isolated adult rat lung, a surface alveolus is micropunctured and a test solution, labeled with a low concentration of fluorescent dye verified not to alter surface tension, is instilled. In flooded alveoli, the air-liquid interface forms a meniscus, at which surface tension is determined as follows. Alveolar air pressure is determined with a transducer at the trachea of the constantly-inflated lung. Alveolar liquid phase pressure is determined by servo-nulling pressure measurement. Meniscus radius of curvature is determined by confocal microscopy. Surface tension is calculated according to the Lapalce relation.
Method 2. Ventilation ‘injury score’ in the adult rat lung. In the isolated, perfused adult rat lung, a surface alveolus is micropunctured and a non-fluorescent test solution is instilled. In experimental regions, a sufficiently large volume of liquid is instilled to generate a pattern of heterogeneous alveolar flooding; in control regions, a sufficiently small volume of liquid is instilled that the liquid spontaneously clears from the region, leaving behind a micropunctured-but-aerated region. Fluorescent dye, at a low concentration verified not to alter surface tension, is included in the perfusate. The region is imaged by confocal microscopy over a five minute baseline period at a constant transpulmonary pressure of 5 cm H2O. Five ventilation cycles are supplied to the lung, at 0.33 Hz with a positive end-expiratory pressure of 15 cm H2O and a tidal volume of 6 ml/kg body weight. The lung is then returned to a constant transpulmonary pressure of 5 cm H2O and imaged for 10 additional minutes. Alveolar liquid fluorescence at all time points is normalized by capillary fluorescence.
At baseline, alveolar liquid fluorescence (in flooded alveoli of experimental regions or in the liquid lining layer of control, aerated regions) is low and constant in all regions. Following ventilation, alveolar liquid fluorescence remains unchanged in aerated regions but continually increases with time in heterogeneously flooded regions. This result indicates that in heterogeneously flooded, but not aerated, regions, ventilation injures the alveolar-capillary barrier, permitting fluorescence to pass from the vascular perfusate to the alveolar liquid, and the injury is sustained over time. The increase above baseline in normalized alveolar liquid fluorescence at the last time point of the experiment is used as an injury score. The injury score, which indicates the rate of increase of normalized fluorescence following ventilation, correlates with surface tension of the test solution.
Method 3. Opening pressure of the immature fetal rat lung. To inflate the initially liquid-filled fetal lung for the first time, the pressure applied at the trachea must be sufficient to overcome a capillary force that is proportional to surface tension of the liquid in the lung. Thus, following instillation of a test solution in the trachea of the immature fetal rat lung, the opening pressure of the lung is indicative of the surface tension of the test solution. At embryonic day 18 or 19 (term=day 22), a fetus is delivered from a pregnant rat by uterotomy. (The normalized phospholipid content of the fetal rat lung on embryonic day 19 is 65% of that at full term.) A test solution (4-5 μl) is placed in the tip of a cannula; the cannula is inserted into the trachea and fixed in place with a suture; and a column of water, behind an air-filled cylinder that is connected to the tracheal cannula, is used to raise tracheal pressure in 10 cm H2O steps. The opening pressure that causes air to flow into the lungs is recorded, and is proportional to test solution surface tension.
Method 4. Surface tension in a liquid drop. In a 3 μl drop of liquid (normal saline+31 μM fluorescein, which does not alter surface tension, for fluid visualization+test solutes), surface tension is determined using the same method as in the adult rat lung (method #1, above). Liquid pressure in the drop is determined by servo-nulling pressure measurement; interfacial radius of curvature is determined by confocal microscopy; and air pressure is known to be atmospheric. Surface tension is calculated according to the Laplace relation and found to be 72±2 mN/m for normal saline, as expected.
Surfactant Therapy Limitations.
As noted above, surfactant therapy is successful in premature neonates but has not reduced mortality in ARDS. Even in neonates, there is room for improvement of surfactant therapy. As also noted above, the fact that high plasma protein concentrations are present in the alveolar liquid of premature neonates suggests that aeration, despite surfactant therapy, is sufficiently heterogeneous that stress concentrations are present and exacerbated by mechanical ventilation, resulting in injury to the alveolar-capillary barrier in NRDS. In translating surfactant therapy from neonates to adults while maintaining the same surfactant dosage per kg of body weight, the quantity of surfactant required becomes excessive. Use of a dilute surfactant would reduce the quantity of surfactant required. Further, there is evidence that dilute surfactant solutions distribute more homogeneously throughout the lungs than do concentrated solutions, which could be beneficial to both neonates and adults.
There are concerns about the use of animal surfactant, which include the possibility that it contains prions, which may cause brain disease. Thus attempts have been made to produce a synthetic surfactant. A synthetic surfactant could be the combination of lipids with one or more recombinant or synthetic surfactant protein. Efforts have focused on recombinant and synthetic forms of SP-C.
Surfactant protein C.
SP-C is a 4.2 kilodalton (kD), 34 amino acid peptide. It has an α-helix and an N-terminal region of undefined conformation. Two cysteine residues in the N-terminal region are palmitoylated. Various forms of recombinant SP-C and synthetic SP-C (sSP-C) have been identified and/or tested as a component of synthetic surfactant, in which the role of the recombinant or synthetic SP-C would be to promote lipid adsorption. One such form is the unpalmitoylated sSP-Css-ion lock (GIPSSPVHLKRLLIVVVVVELIVKVIVGALLMGL (SEQ ID NO. 1)) which is disclosed in U.S. Patent Application Publication No. 2015/0125515, which is hereby incorporated herein by reference. In this peptide, serine residues are substituted for the two cysteines, to avoid cross bridge formation and aggregation in the absence of palmitoylation. Additionally, a glutamine with a negatively charged side chain and a lysine with a positively charged side chain are substituted within the α-helix region at residues 20 and 24, respectively. The oppositely charged side chains, located approximately one turn of the α-helix apart, are thought to attract one another and thus form an ‘ion lock’ that stabilizes the α-helix. Alternatively, also as disclosed in U.S. Patent Application Publication No. 2015/0125515, phenylananine residues may be substituted for the two cysteins, to avoid cross bridge formation and aggregation in the absence of palmitoylation. And leucines may be substituted for valines in the a-helix region. Leucines, with longer side chains than valines, help maintain a-helix integrity.
Novel Findings.
Although, to date, no synthetic surfactant has functioned as well as animal surfactant, Applicants have surprisingly found that low concentrations of surfactant, isolated SP-C or isolated sSP-C, in the presence of albumin, can lower surface tension in the lungs and thereby minimize mechanical ventilation injury to an edematous lung. Applicants have tested 1% SURVANTA® solution in conjunction with albumin and alternative negatively charged solutes; human SP-C isolated from pulmonary alveolar proteinosis patients, with albumin; sSP-Css-ion lock, with albumin; sSP-Css-ion lock-B, a variant of sSP-Css-ion lock with a biotinylated N-terminal, with albumin; and sSP-Cffleuc (GIPFFPVHLKRLKLLLLLLLLILLLILGALLMGL (SEQ ID NO. 2)), in which phenylananine residues are substituted for the two cysteins and leucines are substituted for valines in the α-helix region, with albumin. It is believed that besides albumin, an alternative negatively charged solute, such as fibrinogen or negatively charged 70 kD dextran, would cooperate with low concentrations of isolated SP-C or sSP-C to lower surface tension in the lungs and thereby minimize mechanical ventilation injury to an edematous lung. Based on this finding, it is believed that a recombinant or synthetic SP-C, alone, could constitute a synthetic surfactant that could achieve the aforesaid goal. It is noted that, at 4.2 kD, which is smaller than the 66 kD albumin that passes from capillary to alveolus in ARDS, CPE, and NRDS, isolated SP-C could be delivered intravascularly, potentially increasing either the homogeneity of the therapy throughout the lungs or the matching of the therapy to the edematous regions that require it. The sequence listings for unpalmitoylated sSP-Css-ion lock and sSP-Cff-leuc are presented in Table 1 hereinbelow.